Recombination is a major mechanism to generate the genetic diversity of HIV-1. Recombination occurs during reverse transcription using information in the two RNAs to generate a hybrid DNA. Therefore, phenotypically distinct recombinants are generated from viruses containing two copies of different RNAs. Only cells that are doubly infected can generate virions containing two different RNAs. To better understand interactions within the HIV-1 population, we examined the dynamics of HIV-1 double infection. We have demonstrated that HIV-1 double infection occurs far more frequently than expected from random events, and preference in virus entry plays a key role in nonrandom double infection. We have measured HIV-1 recombination rates and have demonstrated that HIV-1 recombines at an exceedingly high rate, approximately 10-fold higher than that of murine leukemia virus (MLV) and spleen necrosis virus (SNV). We have demonstrated that recombination can occur between two viruses within the same HIV-1 subtypes, between viruses from different HIV-1 subtypes, and between distantly related viruses. We have determined factors that affect recombination and have shown that HIV-1 intersubtype recombinants undergo strong purifying selection. In this project, we hypothesize that reverse transcription is a well-choreographed event in the target cells;multiple viral and host factors can affect this process. We will explore the nucleic acid structure in the reverse transcription complex and the factors that affect the dynamics of reverse transcription and recombination. We will also continue to study recombination and viral fitness, including examining the cause of loss of fitness of the intersubtype recombinants and studying the viral population in the infected individuals. This work will further our understanding of the molecular basis for generating HIV genetic diversity and explore the possible emergence of novel human viruses via recombination. [Corresponds to Hu Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]